Tbx4 and Tbx5 are critical for hindlimb and forelimb development, respectively. The developmental regulation of these transcription factors in forelimbs and hindlimbs of mice will be elucidated, and downstream targets of these genes will be identified. Using comparative genomics and mouse transgenics, a hindlimb enhancer has recently been localized to a 5.5kb region of Tbx4. The minimal portion of this sequence that can direct hindlimb expression in transgenic mice will be identified. Experiments will also be performed to isolate cis-regulatory elements that direct the forelimb expression of Tbx5. Candidate trans-acting factors that may act upon these cis-regulatory elements will be identified using comparative genomics and yeast-one hybrid screens, and it will be established whether these candidate factors are directly involved in the regulation of Tbx4 and Tbx5 limb expression. Finally, genes that are differentially expressed between embryonic forelimbs and hindlimbs will be isolated, and it will be determined whether these genes are direct downstream targets of Tbx4 or Tbx5. Collectively, the identification of both upstream regulators and downstream targets of Tbx4 and Tbx5 will help reveal the precise roles that these genes play in forelimb and hindlimb development.